Human and rat PGC-3, PPAR-gamma coactivations and splice variants thereof

ABSTRACT

A novel gene PGC-3, and its role in regulating the transcriptional activity of PPARγ in adipose tissue. PGC-3 is highly expressed in human white adipose tissue and has utility in the development of new therapeutic agents for use in the treatment of obesity and other related disorders such as non-insulin dependent diabetes mellitus, insulin resistance syndrome, dyslipidemia, and atherosclerosis.

This application is a national stage filing under 35 U.S.C. 371 ofInternational Application No. PCT/GB01/04074, filed Sep. 12, 2001, whichclaims priority from United Kingdom Application No. 0022670.4, filedSep. 15, 2000, the specifications of each of which are incorporated byreference herein. International Application No. PCT/GB01/04074 waspublished under PCT Article 21(2) in English.

This invention relates to the regulation of metabolism and in particularto human genes involved in obesity. The invention further relates toproteins encoded by the genes and to means of regulating theirbiological activity. In addition the invention relates to the use of thegenes and proteins to identify therapeutic agents for controllingobesity and other related disorders such as non-insulin dependentdiabetes mellitus (NIDDM), insulin resistance syndrome, dyslipidemia,and atherosclerosis.

Obesity results from an excessive accumulation of adipose tissue and isa growing public health problem in developed and developing countries.Highly overweight individuals show significant increases in theoccurrence of NIDDM, coronary heart disease, some cancers and digestivediseases.

Current treatment is unsatisfactory and new drugs need to be developed.A major problem is that the mechanisms regulating obesity, and the roleof increased adiposity in the development of metabolic dysfunction areunclear. What is apparent is that obesity results from an imbalancebetween energy intake and expenditure. Energy expenditure can beaffected by alterations in basal metabolism, physical activity andadaptive thermogenesis.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a recentlyidentified member of the peroxisome proliferator-activated receptorfamily of nuclear hormone receptors (Tontonoz et al., Genes Dev. (1994)8, 1224–1234). The expression of this protein is induced very early inthe adipocyte differentiation process and, when expressed ectopically infibroblastic cells, induces adipogenesis in response to activators ofthe receptor. Synthetic and naturally occurring ligands for PPARγ havebeen identified. Thiazolidinediones (TZDs), a class of insulinsensitising agents which are used for the treatment of NIDDM, have beenshown to bind to and activate PPARγ. TZDs promote adipocytedifferentiation of murine and human preadipocytes to mature, fat storingadipocytes. TZD activation of PPARγ has also been shown to regulatetranscription of many adipocyte genes. In addition to the presence of aligand, the activity of PPARγ has been shown to be influenced by thepresence of coactivators and corepressors. When co-expressed in cellsalongside PPARγ these proteins have been shown to greatly increase orrepress the transcriptional activity of PPARγ. Differences in expressionof these coactivators and corepressors between cell types may explainthe observed differences in PPARγ mediated transcriptional activitybetween cells from different tissues.

One such coactivator is PGC-1 (Puigserver et al., Cell (1998) 92,829–839). The expression of this 90 kDa nuclear protein is greatlyincreased in muscle and brown fat of mice upon their exposure to coldtemperatures. Co-expression of PGC-1 with PPARγ has been shown toactivate aspects of the adaptive thermogenic program.

However, PGC-1 is not expressed in white adipose tissue which makes upthe majority of adipose tissue found in humans. The identification of aprotein which regulates the activity of PPARγ in white adipose tissue isthus of great importance in understanding the development of humanobesity.

In the present invention we disclose the cloning and identification ofPGC-3, and its role in regulating the transcriptional activity of PPARγin adipose tissue. PGC-3 is highly expressed in human white adiposetissue and shares sequence homology with PGC-1 in domains known to beresponsible for distinct activity of the protein. Two distinct variantsof PGC-3 have been identified, termed PGC-3a and PGC-3b, which arisefrom alternative splicing of the PGC-3 gene. A further splice varianthas been identified, termed PGC-3c. Full length cDNA and proteinsequences for each of the splice variants are provided. The inventionfurther discloses that PGC-3 has utility in the development of newtherapeutic agents for use in the treatment of obesity and other relateddisorders such as non-insulin dependent diabetes mellitus, insulinresistance syndrome, dyslipidemia, and atherosclerosis. The inventionfurther provides methods for the identification of such therapeuticagents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All publications and patentsreferred to herein are incorporated by reference.

The term “PGC-3” as used herein encompasses both of the splice variants,PGC-3a and PGC-3b, as well as PGC-3c.

According to one aspect of the present invention we provide an isolatedand purified polynucleotide molecule comprising a nucleic acid sequencewhich encodes a polypeptide having at least about 90% homology to amember selected from any one of

(a) (SEQ ID NO:2, SEQ ID NO:2 positions 1–600, SEQ ID NO:2 positions400–1002, and SEQ ID NO:2 positions 200–800)

or

(b) (SEQ ID NO:4, SEQ ID NO:4 positions 1–600, SEQ ID NO:4 positions400–996, and SEQ ID NO:4 positions 200–800)

or

(c) (SEQ ID NO: 8, SEQ ID NO: 8 positions 1–600, SEQ ID NO: 8 positions400–1023, and SEQ ID NO: 8 positions 200–800).

Isolated and purified polynucleotides of the present invention includesequences which comprise the human PGC-3a cDNA sequence set out in SEQID NO:1 and the human PGC-3b cDNA sequence set out in SEQ ID NO:3 andthe human PGC-3c cDNA sequence set out in SEQ ID NO:7.

In addition we have also identified and sequenced a rat clone having ahigh degree of homology to PGC-3 (cf. Example 5). Polynucleotide andpolypeptide molecules based on the rat PGC-3 sequence may be used byanalogy with the human sequences. The rat sequence shows a high degreeof sequence homology (78% sequence identity and rats are thereforeexpected to be useful in animal models of metabolism.

Therefore in a further aspect of the invention we provide an isolatedand purified polynucleotide molecule comprising a nucleic acid sequencewhich encodes a polypeptide having at least about 90% homology to anyone of SEQ ID NO:10, SEQ ID NO:10 positions 1–600, SEQ ID NO: 10positions 400–990, and SEQ ID NO:10 positions 200–800.

In a further aspect of the invention we provide fragments of theisolated and purified polynucleotide molecules of the present invention.By fragments we mean contiguous regions of the polynucleotide moleculeincluding complementary DNA and RNA sequences, starting with shortsequences useful as probes or primers of say about 8–50 bases, such as10–30 bases or 15–35 bases, to longer sequences of up to 50, 100, 200,500 or 1000 bases. Indeed any convenient fragment of the polynucleotidemolecule may be a useful fragment for further research, therapeutic ordiagnostic purposes. Further convenient fragments include those whoseterminii are defined by restriction sites within the molecule of one ormore kinds, such as any combination of Rsa1, Alu1 and Hinf1.

In a further aspect we provide homologues and orthologues of theisolated and purified polynucleotide molecules of the present invention.Preferred homologues and orthologues are polynucleotide molecules whichdisplay greater than 80% sequence homology, conveniently greater than85%, for example 90%, to the PGC-3 cDNA sequences set out in SEQ ID NO:1and SEQ ID NO:3. A homologue may be a polynucleotide molecule from thesame species i.e. a homologous family member, alternatively, thehomologue may be a similar polynucleotide molecule from a differentspecies such as human, useful in developing new therapies for thetreatment of IRS and other related disorders such as NIDDM, obesity andatherosclerosis. By the term orthologue we mean a functionallyequivalent molecule in another species. The full sequences of theindividual homologues and orthologues may be determined usingconventional techniques such as hybridisation, PCR and sequencingtechniques, starting with any convenient part of the sequence set out inSEQ ID NO: 1 or SEQ ID NO:3.

In a further aspect of the invention we provide isolated and purifiedpolynucleotide molecules capable of specifically hybridising to thepolynucleotide molecules of the present invention. By specificallyhybridising we mean that the polynucleotide hybridises by base-pairinteractions, under stringent conditions, to the polynucleotidemolecules of the present invention or to the corresponding complementarysequences. Experimental procedures for hybridisation under stringentconditions are well known to persons skilled in the art. For example,hybridisation filters may be incubated overnight at 42° C. in a solutioncomprising 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH7.6) 5× Denhardt's solution, 10% dextransulphate, and 20 μg/ml denatured salmon sperm DNA; followed by washingthe filters in 0.1×SSC at about 65° C. Hybridisation techniques arethoroughly described in Sambrook J., Fritsch E. F. and Maniatis T.,Molecular Cloning a Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989.

In a further aspect we provide an expression vector comprising apolynucleotide molecule of the present invention.

A variety of mammalian expression vectors may be used to express therecombinant polypeptides of the present invention. Commerciallyavailable mammalian expression vectors which are suitable forrecombinant expression include, pcDNA3 (Invitrogen), pMC1neo(Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224),pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146),pUCTag (ATCC 37460), IZD35 (ATCC 37565), pLXIN, pSIR (CLONTECH), andpIRES-EGFP (CLONTECH).

Baculoviral expression systems may also be used with the presentinvention to produce high yields of biologically active polypeptides.Preferred vectors include the CLONTECH, BacPak™ Baculovirus expressionsystem and protocols which are commercially available (CLONTECH, PaloAlto, Calif.).

Further preferred vectors include vectors for use with the mouseerythroleukemia cell (MEL cell) expression system comprising the humanbeta globin gene locus control region (Davies et al., J. of Pharmacol.and Toxicol. Methods 33, 153–158).

Vectors comprising one or more polynucleotide molecules of the presentinvention may then be purified and introduced into appropriate hostcells. Therefore in a further aspect we provide a transformed host cellcomprising a polynucleotide molecule of the present invention.

The polypeptides of the present invention may be expressed in a varietyof hosts such as bacteria, plant cells, insect cells, fungal cells andhuman and animal cells. Eukaryotic recombinant host cells are especiallypreferred. Examples include yeast, mammalian cells including cell linesof human, bovine, porcine, monkey and rodent origin, and insect cellsincluding Drosophila and silkworm derived cell lines. Cell lines derivedfrom mammalian species which may be used and which are commerciallyavailable include, L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCCCCL 1.2), HEK 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61),3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I(ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

The expression vector may be introduced into host cells to express apolypeptide of the present invention via any one of a number oftechniques including calcium phosphate transformation, DEAE-dextrantransformation, cationic lipid mediated lipofection, electroporation orinfection

The transformed host cells are propagated and cloned, for example bylimiting dilution, and analysed to determine the expression level ofrecombinant polypeptide. Identification of transformed host cells whichexpress a polypeptide of the present invention may be achieved byseveral means including immunological reactivity with antibodiesdescribed herein and/or the detection of biological activity.

Polypeptides of the present invention may be expressed as fusionproteins, for example with one or more additional polypeptide domainsadded to facilitate protein purification. Examples of such additionalpolypeptides include metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilisedmetals (Porath, J., Protein Exp. Purif. 3:263 (1992)), protein A domainsthat allow purification on immobilised immunoglobulin, and the domainutilised in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of cleavable linker sequences suchas Factor XA or enterokinase (Invitrogen, San Diego Calif.) between thepurification domain and the coding region is useful to facilitatepurification. A preferred protein purification system is the CLONTECH,TALON™ nondenaturing protein purification kit for purifying 6×His-tagged proteins under native conditions (CLONTECH, Palo Alto,Calif.).

Therefore in a further aspect we provide a method for producing apolypeptide of the present invention, which method comprises culturing atransformed host cell comprising a polynucleotide of the presentinvention under conditions suitable for the expression of saidpolypeptide.

In a further aspect of the present invention we provide a purifiedpolypeptide comprising the human PGC-3a amino acid sequence set out inSEQ ID NO:2 or a variant of SEQ ID NO:2 having at least about 90%homology to a member selected from (SEQ ID NO:2 positions 1–600, SEQ IDNO:2 positions 400–1002, SEQ ID NO:2 positions 200–800), or abiologically active fragment thereof.

In a further aspect of the present invention we provide a purifiedpolypeptide comprising the human PGC-3b amino acid sequence set out inSEQ ID NO:4 or a variant of SEQ ID NO:4 having at least about 90%homology to a member selected from (SEQ ID NO:4 positions 1–600, SEQ IDNO:4 positions 400–996, SEQ ID NO:4 positions 200–800), or abiologically active fragment thereof.

In a further aspect of the present invention we provide a purifiedpolypeptide comprising the human PGC-3c amino acid sequence set out inSEQ ID NO:8 or a variant of SEQ ID NO:8 having at least about 90%homology to a member selected from (SEQ ID NO:8 positions 1–600, SEQ IDNO:8 positions 400–1023, SEQ ID NO:8 positions 200–800), or abiologically active fragment thereof.

In a further aspect of the present invention we provide a purifiedpolypeptide comprising the rat PGC-3 amino acid sequence set out in SEQID NO:10 or a variant of SEQ ID NO:10 having at least about 90% homologyto a member selected from (SEQ ID NO:10 positions 1–600, SEQ ID NO:10positions 400–990, SEQ ID NO:10 positions 200–800), or a biologicallyactive fragment thereof.

A variant is a polynucleotide or polypeptide which differs from areference polynucleotide or polypeptide, but which retains some of itsessential characteristics. For example, a variant of a PGC-3 polypeptidemay have an amino acid sequence that is different by one or more aminoacid substitutions, deletions and/or additions. The variant may haveconservative changes (amino acid similarity), wherein a substitutedamino acid has similar structural or chemical properties, for example,the replacement of leucine with isoleucine. Alternatively, a variant mayhave nonconservative changes, e.g., replacement of a glycine with atryptophan. Guidance in determining which and how many amino acidresidues may be substituted, inserted or deleted and the effect thiswill have on biological activity may be reasonably inferred from thepresent disclosure by a person skilled in the art and may further befound using computer programs well known in the art, for example,DNAStar software.

Amino acid substitutions may be made, for instance, on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues.Negatively charged amino acids, for example, include aspartic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine; asparagine, glutamine; serine, threonine,phenylalanine, and tyrosine.

Suitable substitutions of amino acids include the use of a chemicallyderivatised residue in place of a non-derivatised residue. D-isomers andother known derivatives may also be substituted for the naturallyoccurring amino acids. See, e.g., U.S. Pat. No. 5,652,369, Amino AcidDerivatives, issued Jul. 29, 1997. Example substitutions are set forthin Table 1.

“Homology” as used in this description is a measure of the similarity oridentity of nucleotide sequences or amino acid sequences. In order tocharacterise the homology, subject sequences are aligned so that thehighest order identity match is obtained. Identity can be calculatedusing published techniques. Computer program methods to determineidentity between two sequences, for example, include DNAStar software(DNAStar Inc., Madison, Wis.); the GCG program package (Devereux, J., etal., Nucleic Acids Research 1984, 12(1):387); and BLASTP, BLASTN, FASTA(Atschul, S. F. et al., J Molec Biol 1990, 215:403). Homology as definedherein is determined conventionally using the well known computerprogram, BESTFIT (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis., 53711). When using BESTFIT or another sequencealignment program to determine the similarity of a particular sequenceto a reference sequence, the parameters are typically set such that thepercentage identity is calculated over the full length of the referencenucleotide sequence or amino acid sequence and that gaps in homology ofup to about 10% of the total number of nucleotides or amino acidresidues in the reference sequence are allowed.

In a further aspect we provide polymorphic variants of thepolynucleotides and polypeptides of the present invention. Polymorphismsare variations in polynucleotide or polypeptide sequences between oneindividual and another. DNA polymorphisms may lead to variations inamino acid sequence and consequently to altered protein structure andfunctional activity. Polymorphisms may also affect mRNA synthesis,maturation, transport and stability. Polymorphisms which do not resultin amino acid changes (silent polymorphisms) or which do not alter anyknown consensus sequences may nevertheless have a biological effect, forexample by altering mRNA folding or stability.

Knowledge of polymorphisms may be used to help identify patients mostsuited to therapy with particular pharmaceutical agents (this is oftentermed “pharmacogenetics”). Pharmacogenetics may also be used inpharmaceutical research to assist the drug selection process.Polymorphisms may be used in mapping the human genome and to elucidatethe genetic component of diseases. The reader is directed to thefollowing references for background details on pharmacogenetics andother uses of polymorphism detection: Linder et al. (1997), ClinicalChemistry, 43, 254; Marshall (1997), Nature Biotechnology, 15, 1249;International Patent Application WO 97/40462, Spectra Biomedical; andSchafer et al. (1998), Nature Biotechnology, 16, 33.

The polypeptides of the present invention may be genetically engineeredin such a way that their interaction with other intracellular andmembrane associated proteins are maintained but their effector functionand biological activity are removed. A polypeptide genetically modifiedin this way is known as a dominant negative mutant. In the constructionof a dominant negative mutant at least one amino acid residue positionat a site required for activity in the native peptide is changed toproduce a peptide which has reduced activity or which is devoid ofdetectable activity. Overexpression of the dominant negative mutant inan appropriate cell type down-regulates the effect of the endogenouspolypeptide, thereby revealing the biological mechanisms involved in thecontrol of metabolism.

Similarly, the polypeptides of the present invention may be geneticallyengineered in such a way that their effector function and biologicalactivity are enhanced. The resultant overactive polypeptide is known asa dominant positive mutant. At least one amino acid residue position ata site required for activity in the native peptide is changed to producea peptide which has enhanced activity. Overexpression of a dominantpositive mutant in an appropriate cell type amplifies the response ofthe endogenous native polypeptide highlighting the regulatory mechanismscontrolling cell metabolism.

Therefore in a further aspect we provide dominant negative and dominantpositive mutants of the polypeptides of the present invention.

Novel sequences disclosed herein, may be used in another embodiment ofthe invention to regulate expression of PGC-3 genes in cells by the useof antisense constructs. For example an antisense expression constructmay be readily constructed using the pREP10 vector (InvitrogenCorporation). Transcripts are expected to modulate translation of thegene in cells transfected with the construct. Antisense transcripts areeffective for modulating translation of the native gene transcript, andare capable of altering the effects (e.g., regulation of tissuephysiology) herein described. Oligonucleotides which are complementaryto and hybridisable with any portion of mRNA disclosed herein arecontemplated for therapeutic use. U.S. Pat. No. 5,639,595,“Identification of Novel Drugs and Reagents”, issued Jun. 17, 1997,wherein methods of identifying oligonucleotide sequences that display invivo activity are thoroughly described, is herein incorporated byreference. Antisense molecules may also be synthesised for use inantisense therapy, using techniques known to persons skilled in the art.These antisense molecules may be DNA, stable derivatives of DNA such asphosphorothioates or methylphosphonates, RNA, stable derivatives of RNAsuch as 2′-O-alkylRNA, or other oligonucleotide mimetics. U.S. Pat. No.5,652,355, “Hybrid Oligonucleotide Phosphorothioates”, issued Jul. 29,1997, and U.S. Pat. No. 5,652,356, “Inverted Chimeric and HybridOligonucleotides”, issued Jul. 29, 1997, which describe the synthesisand effect of physiologically-stable antisense molecules, areincorporated by reference. Antisense molecules may be introduced intocells by microinjection, liposome encapsulation or by expression fromvectors harboring the antisense sequence.

In a further aspect we provide an antibody specific for a polypeptide ofthe present invention.

Antibodies can be prepared using any suitable method, for example,purified polypeptide may be utilised to prepare specific antibodies. Theterm “antibodies” includes polyclonal antibodies, monoclonal antibodies,and the various types of antibody constructs such as for exampleF(ab′)₂, Fab and single chain Fv. Antibodies are defined to bespecifically binding if they bind the antigen with a K_(a) of greaterthan or equal to about 10⁷M⁻¹. Affinity of binding can be determinedusing conventional techniques, for example those described by Scatchardet al., Ann. N.Y. Acad. Sci., 51:660 (1949).

Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice or rats, using procedures that are well-known in the art.In general, antigen is administered to the host animal typically throughparenteral injection. The immunogenicity of antigen may be enhancedthrough the use of an adjuvant, for example, Freund's complete orincomplete adjuvant. Following booster immunisations, small samples ofserum are collected and tested for reactivity to antigen. Examples ofvarious assays useful for such determination include those described in:Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; as well as procedures such ascountercurrent immuno-electrophoresis (CIEP), radioimmunoassay,radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA),dot blot assays, and sandwich assays, see U.S. Pat. Nos. 4,376,110 and4,486,530.

Monoclonal antibodies may be readily prepared using well-knownprocedures, see for example, the procedures described in U.S. Pat. Nos.RE 32,011, 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKearn, and Bechtol (eds.), (1980).

The monoclonal antibodies of the invention can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas”, Strategies in Molecular Biology 3: 1–9 (1990) which isincorporated herein by reference. Similarly, binding partners can beconstructed using recombinant DNA techniques to incorporate the variableregions of a gene that encodes a specific binding antibody. Such atechnique is described in Larrick et al., Biotechnology, 7: 394 (1989).

Once isolated and purified, the antibodies may be used to detect thepresence of antigen in a sample using established assay protocols.

In a further aspect of the invention we provide a method for identifyinga therapeutic agent capable of modulating the activity of PGC-3 for usein the regulation of metabolism, which method comprises:

(i) contacting a candidate compound modulator with a PGC-3 polypeptidecomprising any one of

(a) the amino acid sequence set out in SEQ ID NO:2 or a variant of SEQID NO:2 having at least about 90% homology to a member selected from(SEQ ID NO:2 positions 1–600, SEQ ID NO:2 positions 400–1002, SEQ IDNO:2 positions 200–800) or a biologically active fragment thereof;or(b) the amino acid sequence set out in SEQ ID NO:4 or a variant of SEQID NO:4 having at least about 90% homology to a member selected from(SEQ ID NO:4 positions 1–600, SEQ ID NO:4 positions 400–996, SEQ ID NO:4positions 200–800) or a biologically active fragment thereof;or(c) the amino acid sequence set out in SEQ ID NO:8 or a variant of SEQID NO:8 having at least about 90% homology to a member selected from(SEQ ID NO:8 positions 1–600, SEQ ID NO:8 positions 400–996, SEQ ID NO:8positions 200–800) or a biologically active fragment thereof;and(ii) measuring an effect of the candidate compound modulator on theactivity of the PGC-3 polypeptide.

Activity as used herein refers to the ability of the therapeutic agentto mediate cell processes related to insulin resistance syndrome andother related disorders such as non-insulin dependent diabetes mellitus,dyslipidemia, obesity and atherosclerosis.

Modulation of the activity of PGC-3 comprises either stimulation orinhibition. Thus a therapeutic agent capable of modulating the activityof PGC-3 is an agent that either stimulates or inhibits the activity ofPGC-3. The terms “modulator of PGC-3 activity” and “PGC-3 modulator” arealso used herein to refer to an agent that either stimulates or inhibitsthe activity of PGC-3. The therapeutic agents of the invention haveutility in the regulation of metabolism; in particular in obesity andthe control of insulin resistance syndrome and other related disorderssuch as non-insulin dependent diabetes mellitus, dyslipidemia, andatherosclerosis.

In a further aspect of the invention we provide a screen for identifyingcompounds which modulate the activity of PGC-3, the invention extends tosuch a screen and to the use of compounds obtainable therefrom tomodulate the activity of PGC-3 in vivo.

Potential therapeutic agents which may be tested in the screen includesimple organic molecules, commonly known as “small molecules”, forexample those having a molecular weight of less than 2000 Daltons. Thescreen may also be used to screen compound libraries such as peptidelibraries, including synthetic peptide libraries and peptide phagelibraries. Other suitable molecules include antibodies, nucleotidesequences and any other molecules which modulate the activity of PGC-3.

Once an inhibitor or stimulator of PGC-3 activity is identified thenmedicinal chemistry techniques can be applied to further refine itsproperties, for example to enhance efficacy and/or reduce side effects.

It will be appreciated that there are many screening procedures whichmay be employed to perform the present invention. Examples of suitablescreening procedures which may be used to identify a PGC-3 modulator foruse in the regulation of metabolism include rapid filtration ofequilibrium binding mixtures, enzyme linked immunosorbent assays(ELISA), radioimmunoassays (RIA) and fluorescence resonance energytransfer assays (FRET). For further information on FRET the reader isdirected to International Patent Application WO 94/28166 (Zeneca).Methods to identify potential drug candidates have been reviewed byBevan P et al., 1995, TIBTECH 13 115.

A preferred method for identifying a compound capable of modulating theactivity of PGC-3 is a scintillation proximity assay (SPA). SPA involvesthe use of fluomicrospheres coated with acceptor molecules, such asreceptors, to which a ligand will bind selectively in a reversiblemanner (N Bosworth & P Towers, Nature, 341, 167–168, 1989). Thetechnique requires the use of a ligand labelled with an isotope thatemits low energy radiation which is dissipated easily into an aqueousmedium. At any point during an assay, bound labelled ligands will be inclose proximity to the fluomicrospheres, allowing the emitted energy toactivate the fluor and produce light. In contrast, the vast majority ofunbound labelled ligands will be too far from the fluomicrospheres toenable the transfer of energy. Bound ligands produce light but freeligands do not, allowing the extent of ligand binding to be measuredwithout the need to separate bound and free ligand.

Cellular assay systems may be used to further identify PGC-3 modulatorsfor use in the regulation of metabolism.

Therefore in a further aspect of the invention the candidate compoundmodulator is contacted with a host-cell which expresses an PGC-3polypeptide (as hereindefined).

A preferred cellular assay system for use in the method of the inventionis a two-hybrid assay system. The two-hybrid system utilises the abilityof a pair of interacting proteins to bring the activation domain of atranscription factor into close proximity with its DNA-binding domain,restoring the functional activity of the transcription factor andinducing the expression of a reporter gene (S Fields & 0 Song, Nature,340, 245–246, 1989). Commercially available systems such as the ClontechMatchmaker™ systems and protocols may be used with the presentinvention.

Other preferred cellular assay systems include measurement of changes inthe levels of intracellular signalling molecules such as cyclic-AMP,intracellular calcium ions, or arachidonic acid metabolite release.These may all be measured using standard published procedures andcommercially available reagents. In addition the polynucleotides of thepresent invention may be transfected into appropriate cell lines thathave been transfected with a “reporter” gene such as bacterial lacZ,luciferase, aequorin or green fluorescent protein that will “report”these intracellular changes (Egerton et al, J. Mol, Endocrinol, 1995,14(2), 179–189).

In a further aspect of the present invention we provide a novel PGC-3modulator, or a pharmaceutically acceptable salt thereof, for use in amethod of treatment of metabolic diseases of the human or animal body bytherapy.

Examples of metabolic diseases which may be treated using a compound ofthe invention include insulin resistance syndrome, non-insulin dependentdiabetes mellitus, dyslipidemia, obesity and atherosclerosis.

According to a further aspect of the invention, we provide apharmaceutical composition which comprises a novel PGC-3 modulator, or apharmaceutically acceptable salt thereof, in association with apharmaceutically acceptable diluent or carrier.

The composition may be in the form suitable for oral use, for example atablet, capsule, aqueous or oily solution, suspension or emulsion; fortopical use, for example a cream, ointment, gel or an aqueous or oilysolution or suspension; for nasal use, for example a snuff, nasal sprayor nasal drops; for rectal use, for example a suppository; foradministration by inhalation, for example as a finely divided powdersuch as a dry powder, a microcrystalline form or a liquid aerosol; forsub-lingual or buccal use, for example a tablet or capsule; or forparenteral use (including intravenous, subcutaneous, intramuscular,intravascular or infusion), for example a sterile aqueous or oilysolution or suspension. In general, the above compositions may beprepared in a conventional manner using conventional excipients.

The invention also provides a method of treating a metabolic disease ormedical condition mediated alone or in part by PGC-3, which comprisesadministering to a warm-blooded animal requiring such treatment aneffective amount of an PGC-3 modulator as defined above.

The invention also provides the use of an PGC-3 modulator in theproduction of a medicament for use in the treatment of a metabolicdisease.

The amount of active ingredient that is combined with one or moreexcipients to produce a single dosage form will necessarily varydepending on the subject treated and the particular route ofadministration. For example, a formulation intended for oraladministration to humans will generally contain for example, from 0.5 mgto 2 g of active agent compounded with an appropriate and convenientamount of excipients which may vary from about 5 to about 98 percent byweight of the total composition. Dosage unit forms will generallycontain about 1 mg to about 500 mg of an active ingredient.

The size of the dose for therapeutic or prophylactic purposes of anPGC-3 modulator will naturally vary according to the nature and severityof the immune disease, the age and sex of the patient, and the route ofadministration, according to well known principles of medicine.

In using an PGC-3 modulator for therapeutic or prophylactic purposes itwill generally be administered so that a daily dose in the range forexample 0.5 mg to 75 mg per kg body weight is received, given ifrequired in divided doses. In general lower doses will be administeredwhen a parenteral route is employed. Thus for example, for intravenousadministration, a dose in the range for example 0.5 mg to 30 mg per kgbody weight will generally be used. Similarly, for administration byinhalation a dose in the range for example 0.5 mg to 25 mg per kg bodyweight will be used.

The invention will now be illustrated but not limited by reference tothe following Tables, Examples and Figures. Unless indicated otherwise,the techniques used are those detailed in well known molecular biologytextbooks such as Sambrook, Fritsch & Maniatis, Molecular Cloning aLaboratory Manual, second edition, 1989, Cold Spring arbor LaboratoryPress.

SEQ ID NO:1. shows the full length human PGC-3a cDNA

SEQ ID NO:2. shows human PGC-3a protein sequence

SEQ ID NO:3. shows human PGC-3b cDNA

SEQ ID NO:4. shows human PGC-3b protein sequence

SEQ ID NO:5. shows the sequence of the 3′ RACE product isolated fromhuman adipocyte cDNA.

SEQ ID NO:6. shows the sequence of the 5′RACE product isolated fromhuman heart cDNA.

SEQ ID NO:7. shows human PGC-3c cDNA

SEQ ID NO 8. shows human PGC-3c protein sequence

SEQ ID NO 9. shows the full length rat PGC-3 cDNA

SEQ ID NO 10. shows rat PGC-3 protein sequence

FIGURE LEGENDS

FIG. 1 shows specific PCR products of 561 bp for PGC-3b and 491 bp forPGC-3a, isolated from breast adipose tissue cDNA, in lanes 1 and 2respectively. The PGC-3b specific PCR product was obtained using PCRprimers CME9830 and CME9831 (Table 2). The PGC-3a specific PCR productwas obtained using PCR primers CME9830 and CME9850 (Table 2).

FIG. 2 shows a comparison of PGC-3a and PGC-3b with PGC-1, indicatingthat the molecules share regions of sequence homology in particularlocations which are believed to be important for biological activity.

FIG. 3 shows a comparison of human PGC-3a (SEQ ID NO: 2) and rat PGC-3(SEQ ID NO: 10) protein sequences indicating that the molecules share ahigh degree of sequence homology.

FIG. 4 shows the mean relative expression of PGC-3 mRNA in a range ofhuman tissues. Quantitative real-time PCR was undertaken inquadruplicate on cDNA samples derived from the human tissues listedusing Taqman™ fluorescent PCR technology (PE. Applied Biosystems). Thenormalised ratio of expression of PGC-3 relative to a housekeeping gene(GAPDH) was calculated for all tissues. SC=subcutaneous. Where multiplesamples of the same tissue type were assayed the sample number is given,eg Omental adipocyte S24 refers to the data obtained from the omentaladipocyte sample number 24. AU=arbitrary units.

TABLES

TABLE 1 Examples of conservative amino acid substitutions Originalresidue Example conservative substitutions Ala (A) Gly; Ser; Val; Leu;Ile; Pro Arg (R) Lys; His; Gln; Asn Asn (N) Gln; His; Lys; Arg Asp (D)Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro His (H) Asn;Gln; Arg; Lys Ile (I) Leu; Val; Met; Ala; Phe Leu (L) Ile; Val; Met;Ala; Phe Lys (K) Arg; Gln; His; Asn Met (M) Leu; Tyr; Ile; Phe Phe (F)Met; Leu; Tyr; Val; Ile; Ala Pro (P) Ala; Gly Ser (S) Thr Thr (T) SerTrp (W) Tyr; Phe Tyr (Y) Trp; Phe; Thr; Ser Val (V) Ile; Leu; Met; Phe;Ala

TABLE 2 Primer sequences Primer Sequence forward primer CME 97485′ GTCACAAAGCGACCCAACTT 3′ (SEQ ID NO: 11) reverse primer CME 97495′ GAGTCATGGTCTCCAAAGGAAC 3′ (SEQ ID NO: 12) AP1 adaptor primer5′ CCATCCTAATACGACTCACTATAGGGC 3′ (SEQ ID NO: 13) CME 9830 forwardprimer 5′ GCCACTCGAAGGAACTTCAGAT 3′ (SEQ ID NO: 14) CME 9850 reverseprimer B 5′ GGGTTAAGGCTGTTATCAATGC 3′ (SEQ ID NO: 15) CME 9831 reverseprimer A 5′ AGGCCAGAAGAGAAACAGGATG 3′ (SEQ ID NO: 16) CME 9726sequencing primer 5′ CTTCTCCTGTTCCTTTGGAGAC 3′ (SEQ ID NO: 17) CME 9727sequencing primer 5′ TGGGGTTCACTTGAGGATTG 3′ (SEQ ID NO: 18) CME 9778sequencing primer 5′ ATTCAAAATCTCTTCCAGCGAC 3′ (SEQ ID NO: 19) CME 9776sequencing primer 5′ GAAGACAGAAGCTGTGATGCTG 3′ (SEQ ID NO: 20)

TABLE 3 Primers used in Example 4 Primer Sequence SP1A5′-CATCACAGAGCACGTCTTGAG-3′ (SEQ ID NO: 21) SP2A5′-CATGTAGCGTATGAGTTGCACCATC-3′ (SEQ ID NO: 22) Oligo d(T)-anchor5′-GACCACGCGTATCGATGTCGACTTTTT primer TTTTTTTTTTTV-3′ V = A, C or G (SEQID NO: 23) PCR anchor primer 5′-GACCACGCGTATCGATGTCGAC-3 (SEQ ID NO: 24)

TABLE 4 Details of primers used to sequence rat PGC-3 Primer primersequence (5′→3′) CVGI169 TTGGGTAACGCCAGGGTTTTCCCAGTCAC (SEQ ID NO: 25)CVGI170 CCCCAGGCTTTACACTTTATGCTTCCGGC (SEQ ID NO: 26) CVGI171GCCAGTACAGCCCTGATGAT (SEQ ID NO: 27) CVGI172 TCCCCAGTGTCTGAAGTGGATG (SEQID NO: 28) CVGI281 CTCATTCGCTACATGCATACCT (SEQ ID NO: 29) CVGI282CGGCCTTGTGTCAAGGTGGATG (SEQ ID NO: 30) CVGI283 CTTCTGGACTGAGTTCTCCATC(SEQ ID NO: 31) CVGI390 CAGGAGACTGAATCCAGAGCTG (SEQ ID NO: 32) CVGI391GACAGTAGTCAAGGCCAGCAGC (SEQ ID NO: 33) CVGI457 GAGACCATGACTACTGCCAGGT(SEQ ID NO: 34) CVGI458 ACCGCTCTGGAGGAGGAAGACT (SEQ ID NO: 35) CVGI535TTAAGCCTTAACCCTTTGAGGA (SEQ ID NO: 36) CVGI536 GGCCCAGATACACCGACTATGA(SEQ ID NO: 37)

EXAMPLES Example 1

Isolation of Partial PGC-3 cDNA

Method:

PCR primers CME 9748 and CME 9749, listed in Table 2 were synthesisedand used to amplify a 347 bp product from human adipose cDNA (Humanadipocyte Marathon Ready cDNA, Clontech Cat.#7447-1, Clontech,Basingstoke, UK).

A technique known as Rapid Amplification of cDNA Ends (RACE) was thenused to amplify the 3′ end of the PGC-3 cDNA. RACE is a commonly usedmolecular biological technique which enables the user to extend andidentify sequence along a cDNA template in one direction. This allowsthe user to obtain a complete cDNA sequence starting from a small pieceof cDNA sequence. For a more complete description of the method refer toChenchick A, Moqadam F and Siebert P. 1996 Laboratory guide to RNA:isolation, analysis and synthesis. Wiley-Liss Inc. p 273–321. In thiscase we used a commercially available RACE PCR kit, the Human adipocyteMarathon Ready™ cDNA (Clontech, Basingstoke, UK). It is a premade humanadipocyte “library” of adaptor-ligated double stranded cDNA ready forperforming both 5′ and 3′ RACE from the same template. The PGC-3 genespecific primer CME 9748 (Table 2) was used in a Marathon RACE reactionwith the AP1 adapter primer (Table 2) supplied by Clontech and theMarathon Ready™ cDNA according to the manufacturer's instructions.

Results:

A 1.5 kb PCR product was amplified in the reaction and separated fromnon-specific DNA by agarose gel electrophoresis using a 1.5% agarose geland visualised by ethidium bromide staining. The 1.5 kb PCR product wasisolated from the gel using a DNA extraction kit (Qiaex II™, Qiagen) andthe purified PCR product cloned into PCR2.1™ vector using a TOPO TA™Cloning kit (Invitrogen), according to the manufacturer's instructions.The cloned PCR product was fully sequenced using the vector M13sequencing primers supplied in the kit (Invitrogen) and the PGC-3 genespecific sequencing primers CME 9726, CME 9727, CME 9778 and CME 9776(listed in Table 2). The sequence of the 3′RACE product is shown in FIG.5 (SEQ ID NO: 5). The predicted protein sequence of the 3′ end of SEQ IDNO:7 was found to be 50% identical to the sequence for human PGC-1 overa 135 aa region (see FIG. 7). This small region of SEQ ID NO:7 alsoshared 53% identity to the rat PGC-1 sequence (EMBL accession number:AB025784).

Two variants of PGC-3 have been found with cDNA sequence which differ atthe 3′ end. We have named these two variants PGC-3a and PGC-3b.

Example 2

Isolation of a Full Length PGC-3a Clone from a Human Heart cDNA Library.

Method:

Primers CME 9830 and CME 9850 (Table 2) were synthesised based on thePGC-3a 3′ RACE product sequence. These were used to PCR screen theOrigene human heart cDNA library master plate (ORIGENE LHT-1001,Origene, USA) according to the manufacturers instructions.

Results:

PCR screening of the master plate identified several wells positive forPGC-3a cDNA. The subplates corresponding to these wells were obtainedfrom Origene and a subsequent round of PCR screening was performed toidentify individual clones containing PGC-3a cDNA.

Clones containing PGC-3a were identified and sequenced. This resulted inthe isolation of the complete cDNA sequence for PGC-3a (SEQ ID NO:1).The PGC-3a cDNA sequence comprises a coding region of 3009 nucleotides,that encode a protein of 1002 amino acids with a calculated molecularmass of 110 kDa and an estimated isoelectric point of 4.933. The proteinsequence for PGC-3a is shown in SEQ ID NO:2.

Example 3

PCR Amplification of PGC-3a and PGC-3b from Human Breast Adipocyte cDNA

Methods:

PCR primers were synthesised which would specifically amplify eitherPGC-3a or PGC-3b (CME 9830, CME 9831, CME 9850, Table 2).

To investigate whether both PGC-3a and PGC-3b were expressed by humanbreast adipocytes, PCRs were carried out using the above primers toamplify PGC-3a and PGC-3b from human breast adipocyte cDNA. The PCRconditions used were 94° C. for 1 minute then 30 cycles of 94° C. for 30sec, then 68° C. for 4 minutes. The DNA polymerase used was Extensor™from Advanced Biotechnologies. PCR was performed according to standardprocedure described in Molecular Cloning, a laboratory manual, Sambrook,Fritsch and Maniatis Second Ed 1989). The forward PCR primer (CME 9830)was used in combination with either reverse PCR primer A (CME 9831)designed specifically to amplify PGC-3b (sequence 2) or reverse primer B(CME 9850) designed specifically to amplify sequence 3 PGC-3a (3′ RACEproduct).

Results:

Specific PCR products of 561 bp for PGC-3b (CME9830/CME 9831) and 491 bpfor PGC-3a (CME9830/CME9850) were obtained, as shown in FIG. 1 in lanes1 and 2 respectively.

The complete cDNA sequence for PGC-3b is shown in SEQ ID NO:3. ThePGC-3b cDNA sequence comprises a coding region of 2991 nucleotidesencoding a protein of 996 amino acids. The protein sequence for PGC-3bis shown in SEQ ID NO:4.

Example 4

Isolation of Partial PGC-3c cDNA

The technique known as RACE as described in example 1 was used toamplify the 5′ end of the PGC-3c cDNA. This procedure was undertakenusing the Roche 5′/3′RACE kit (Cat. No. 1 734 792) and followed themanufacturer's instructions. First strand cDNA was synthesized fromtotal human heart RNA (Stratagene, Cat. No. 735012-41) using a genespecific primer (SP1A, listed in Table 3), AMV reverse transcriptase(supplied in kit) and deoxynucleotide mix (supplied in kit). The firststrand cDNA was purified using High Pure PCR Product Purification Kit(Roche Diagnostics Corporation, Indianapolis, Ind., USA—Cat No. 1 732668) according to the manufacturer's instructions. A homopolymericA-tail was then added to the 3′end of the cDNA using terminaletransferase using reagents and instructions supplied with the kit. ThecDNA was then amplified by PCR using a gene specific primer (SP2A, seetable 3) and an oligo dT-anchor primer (see table 3). The obtained cDNAwas further amplified by a second PCR using a nested specific primer(SP3A, see Table 3) and a PCR anchor primer (see table 3). Resulting5′RACE products were cloned into a vector. The cloned PCR products werefully sequenced. The sequence of the 5′RACE product is shown in FigureSEQ ID NO: 6. The full length cDNA sequence of PGC-3c is shown in SEQ IDNO: 7. The predicted protein sequence of PGC-3c is shown in SEQ ID NO:8.

Example 5

Isolation of Full Length Rat PGC-3 cDNA

Homology searching using the human PGC-3 cDNA sequence identified a ratclone in a proprietary database that had a high level of homology toPGC-3. This clone was obtained and sequenced using primers CVGI169,CVGI170, CVGI171, CVGI172, CVGI281, CVGI282, CVGI283, CVGI390, CVGI391,CVGI457, CVGI458, CVGI535 (see table 4 for sequence information). Thefull length rat PGC-3 cDNA sequence is shown in SEQ ID NO: 9. Thepredicted protein sequence of rat PGC-3 is shown in Figure SEQ ID NO:10. FIG. 13 shows a comparison of human PGC-3a and rat PGC-3 proteinsequences, indicating that the molecules have a high degree of sequencehomology (greater than 78% identity).

Example 6

Comparison of Human PGC-3 mRNA Expression Between Tissues

Total RNA was extracted from human adipocytes using TR1 reagent(Sigma-Aldrich) following the manufacturer's suggested protocol. Twomicrograms of total RNA from each adipocyte samples was used to generatecDNA, using the Promega reverse transcription system (Promega; cataloguenumber A3500) according to the manufacturer's instructions. The heart,skeletal muscle, kidney, liver, lung, heart and pancreas cDNAs wereobtained from Clontech (Clontech, catalogue numbers K1420-1 andK1421-1). Probe and primer sequences were designed for PGC-3 and were:PGC-3 forward primer; 5′-TGCTGGCCCAGATACACTGA-3′ (SEQ ID NO: 38). PGC-3reverse primer; 5′-GGCTGTTATCAATGCAGGCTC-3′ (SEQ ID NO: 39). PGC-3probe; 5-FAM-CGTCAGGGAAAAGCAAGTATGAAGCCAT-TAMRA-3′ (SEQ ID NO: 40).Taqman PCR assays for each target gene were performed in quadruplicatein 96 well plates on an ABI

1. An isolated and purified polynucleotide comprising a nucleic acidsequence which encodes a polypeptide having at least about 90% homologyto a member selected from any one of: (a) SEQ ID NO: 2, SEQ ID NO: 2positions 1–600, SEQ ID NO: 2 positions 400–1002, and SEQ ID NO: 2positions 200–800, or (b) SEQ ID NO: 4, SEQ ID NO: 4 positions 1–600,SEQ ID NO: 4 positions 400–996, and SEQ ID NO: 4 positions 200–800, or(c) SEQ ID NO: 8, SEQ ID NO: 8 positions 1–600, SEQ ID NO: 8 positions400–1023, and SEQ ID NO: 8 positions 200–800, wherein said polypeptideregulates the transcriptional activity of PPAR-γ.
 2. A polynucleotidewhich comprises the human PGC-3a cDNA sequence set out in SEQ ID NO: 1.3. A polynucleotide which comprises the human PGC-3b cDNA sequence setout in SEQ ID NO:
 3. 4. A polynucleotide which comprises the humanPGC-3c cDNA sequence set out in SEQ ID NO:
 7. 5. An expression vectorcomprising a polynucleotide according to any of claims 1–4.
 6. Atransformed host cell comprising a polynucleotide according to any ofclaims 1–4.
 7. An isolated and purified polynucleotide moleculecomprising a nucleic acid sequence which encodes a polypeptide having atleast about 90% homology to any one of SEQ ID NO: 10 positions 1–600,SEQ ID NO: 10 positions 400–990, and SEQ ID NO: 10 positions 200–800,wherein said polypeptide regulates the transcriptional activity ofPPAR-γ.
 8. A purified polypeptide comprising the human PGC-3a amino acidsequence set out in SEQ D NO: 2; or a variant of SEQ ID NO: 2 having atleast about 90% homology to a member selected from: SEQ ID NO: 2positions 1–600, SEQ ID NO: 2 positions 400–1002, or SEQ ID NO: 2positions 200–800, wherein said variant regulates the transcriptionalactivity of PPAR-γ, or a biologically active fragment thereof, whereinthe fragment regulates the transcriptional activity of PPAR-γ.
 9. Apurified polypeptide comprising the human PGC-3b amino acid sequence setout in SEQ D NO: 4; or a variant of SEQ ID NO: 4 having at least about90% homology to a member selected from: SEQ ID NO: 4 positions 1–600,SEQ ID NO: 4 positions 400–996, or SEQ ID NO: 4 positions 200–800,wherein said variant regulates the transcriptional activity of PPAR-γ,or a biologically active fragment thereof, wherein the fragmentregulates the transcriptional activity of PPAR-γ.
 10. A purifiedpolypeptide comprising the human PGC-3c amino acid sequence set out inSEQ ID NO: 8; or a variant of SEQ ID NO: 8 having at least about 90%homology to a member selected from: SEQ ID NO: 8 positions 1–600, SEQ IDNO: 8 positions 400–1023, or SEQ ID NO: 8 positions 200–800, whereinsaid variant regulates the transcriptional activity of PPAR-γ, or abiologically active fragment thereof, wherein the fragment regulates thetranscriptional activity of PPAR-γ.
 11. A purified polypeptidecomprising the rat PGC-3 amino acid sequence set out in SEQ ID NO: 10;or a variant of SEQ ID NO: 10 having at least about 90% homology to amember selected from: SEQ ID NO; 10 positions 1–600, SEQ ID NO: 10positions 400–990, or SEQ ID NO: 10 positions 200–800, wherein saidvariant regulates the transcriptional activity of PPAR-γ, or abiologically active fragment thereof, wherein the fragment regulates thetranscriptional activity of PPAR-γ.
 12. A method for identifying atherapeutic agent capable of modulating the activity of PGC-3 for use inthe regulation of metabolism, which method comprises: (i) contacting acandidate compound modulator with a PGC-3 polypeptide comprising any oneof (a) the amino acid sequence set out in SEQ ID NO: 2; or a variant ofSEQ ID NO: 2 having at least about 90% homology to a member selectedfrom; SEQ ID NO: 2 positions 1–600, SEQ ID NO: 2 positions 400–1002, orSEQ ID NO: 2 positions 200–800, wherein said variant regulates thetranscriptional activity of PPAR-γ, or a biologically active fragmentthereof, wherein the fragment regulates the transcriptional activity ofPPAR-γ, or (b) the amino acid sequence set out in SEQ ID NO: 4; or avariant of SEQ ID NO: 4 having at least about 90% homology to a memberselected from: SEQ ID NO: 4 positions 1–600, SEQ ID NO: 4 positions400–996, or SEQ ID NO: 4 positions 200–800, wherein said variantregulates the transcriptional activity of PPAR-γ, or a biologicallyactive fragment thereof, wherein the fragment regulates thetranscriptional activity of PPAR-γ, or (c) the amino acid sequence setout in SEQ ID NO: 8; or a variant of SEQ ID NO: 8 having at least about90% homology to a member selected from: SEQ ID NO: 8 positions 1–600,SEQ ID NO: 8 positions 400–996, or SEQ ID NO: 8 positions 200–800,wherein said variant regulates the transcriptional activity of PPAR-γ;or a biologically active fragment thereof, wherein the fragmentregulates the transcriptional activity of PPAR-γ, and (ii) measuring aneffect of the candidate compound modulator on the activity of the PGC-3polypeptide.
 13. A method as claimed in claim 12, wherein the candidatecompound modulator is contacted with a host-cell which expresses a PGC-3polypeptide.